insects Article Evaluation of Drimys winteri (Canelo) Essential Oil as Insecticide against Acanthoscelides obtectus (Coleoptera: Bruchidae) and Aegorhinus superciliosus (Coleoptera: Curculionidae) Jocelyne Tampe 1,2,* , Javier Espinoza 3,4 , Manuel Chacón-Fuentes 3,4, Andrés Quiroz 3,4 and Mónica Rubilar 1,2,* 1 Technology and Processes Laboratory, Department of Chemical Engineering, Faculty of Engineering and Science, Universidad de La Frontera, Avenida Francisco Salazar 01145, Temuco 4811230, Chile 2 Scientific and Technological Bioresource Nucleus, BIOREN, Universidad de La Frontera, Avenida Francisco Salazar 01145, Temuco 4811230, Chile 3 Laboratorio de Química Ecológica, Departamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Casilla 54-D, Avenida Francisco Salazar 01145, Temuco 4811230, Chile; [email protected] (J.E.); [email protected] (M.C.-F.); [email protected] (A.Q.) 4 Centro de Excelencia en Investigación Biotecnológica Aplicada al Medio Ambiente (CIBAMA), Facultad de Ingeniería y Ciencias, Universidad de La Frontera, Avenida Francisco Salazar 01145, Temuco 4811230, Chile * Correspondence: [email protected] (J.T.); [email protected] (M.R.) Received: 22 April 2020; Accepted: 27 May 2020; Published: 30 May 2020 Abstract: Adverse effects caused by synthetic pesticides have increased interest in plant-derived insecticidal compounds, in particular essential oils, as a more compatible and ecofriendly alternative for pest control of economic importance. For this reason, the essential oil isolated from leaves and shoots of Drimys winteri (J.R. Forster & G. Forster)—also named canelo (CEO)—was investigated for its chemical profile and insecticidal action against Acanthoscelides obtectus (Say)—one of the most important post-harvest pests of dry beans in the world—and Aegorhinus superciliosus (Guérin)—a significant pest of fruit trees in Chile. The analysis by gas chromatography, paired with mass spectrometry (GC/MS) determined 56 compounds, corresponding to 92.28% of the detected compounds. Elemol (13.54%), γ-eudesmol (11.42%), β-eudesmol (8.49%), α-eudesmol (6.39%), α-pinene (7.92%) and β-pinene (5.17%) were the most abundant. Regarding the bioactivity of the CEO, the results demonstrated toxicological 1 effects against A. obtectus. A concentration of 158.3 µLL− had a mortality rate of 94% after 24 h 1 exposure. The LC50 and LC90 values at 24 h were 60.1 and 163.0 µLL− . Moreover, behavioral bioassays showed a repellent effect against A. superciliosus with a dose of one microliter of CEO. Both sexes of the raspberry weevil stayed for very short times in the treated area with the oil (<0.8 min), showing a homogeneous repellency in the species. The overall data suggest that canelo leaves and shoots essential oil has an insecticide effect and is worth exploring to better understand the synergistic relationship between the compounds present in the essential oil. Keywords: bean weevil; raspberry weevil; toxic effect; repellent effect 1. Introduction Currently, the study and use of essential oils (EOs) is an interesting and potential tool to develop botanical insecticides safer for health and the environment [1,2]. They are biosynthesized in aromatic plants as secondary metabolites and they play a protective role against biotic factors, such as bacteria, fungi, viruses and insects [1–4]. EOs are composed of a wide variety of volatile Insects 2020, 11, 335; doi:10.3390/insects11060335 www.mdpi.com/journal/insects Insects 2020, 11, 335 2 of 15 compounds, mainly terpenes at high concentrations (between 20%–70%) in addition to other chemical groups present in trace amounts [1]. In overall, the bioactivity of EOs has been recorded to act by contact, ingestion and as fumigants as well as by their antifeedant or repellent action on different pest insects [5–7]. In this context, recent investigations have showed the insecticidal activity of different EOs to manage Prostephanus truncatus (Horn) (Coleoptera: Bostrychidae) and Trogoderma granarium (Everts) (Coleoptera: Dermestidae) infestations [8] against different flies; Calliphora vomitoria (L.) (Diptera: Calliphoridae) [9], Ceratitis capitata (Wiedemann) and Anastrepha fraterculus (Wiedemann) (Diptera: Tephritidae) [10] against Callosobruchus maculatus (Fabricius) (Coleoptera: Chrysomelidae), the main cowpea pest [11] and against two lepidopteran pests, Spodoptera frugiperda (Walker) and Anticarsia gemmatalis (Hübner) (Lepidoptera: Noctuidae) [12], proving to be a useful and effective tool in reducing harmful insect populations and the use of conventional pesticides [13]. Drimys winteri (J.R. Forster & G. Forster) (Winteraceae)—commonly known as canelo, voigue or boighe—is a perennial tree growing in the sub-Antarctic forests of Chile and Argentina [14]. This species is characterized as having great ecological plasticity [15], growing in wetlands and marshes [16] and even in lands left bare due to fire [17]. It is described as a tree with a thick and soft bark, reaching up to 30 m height, with large, ovate very aromatic and smooth leaves [18]. In Chilean indigenous culture, canelo is known as a medicinal plant [18,19], and has been widely used by the Mapuche people as a sacred tree, as it symbolizes the Axis Mundi [20], in addition to its healing, disinfectant and antibacterial properties [21,22]. Canelo bioactivity has been reported from its compounds and EOs obtained from its stems, leaves and bark. Phytochemical studies have shown the activity of polygodial and drimenol sesquiterpenes for medical applications [23], as well as pest control [24,25]. Moreover, the D. winteri EO has produced insecticidal activity against aphids [26], stored grain insects [27] and weevils [28], showing great potential as a natural pesticide. However, there are few the studies that report the insecticidal potential of D. winteri EO. Therefore, our work seeks to expand the knowledge of canelo insecticidal bioactivity towards two important pests of the order Coleoptera—the bean weevil A. obtectus, and the raspberry weevil A. superciliosus. The bean weevil, Acanthoscelides obtectus (Say) (Coleoptera: Bruchidae), is one of the world’s most important post-harvest pests in dry beans, Phaseolus vulgaris (L.) (Fabaceae) [29,30]. It is mainly found in South America, Africa and the Mediterranean [31–33], where the adult attacks bean seeds while they are still in the field and continues to cause damage during storage. This can cause the total loss of stored bean seeds [34,35] as the larvae enter the bean seeds to feed and develop from larva to adult inside the seeds [36]. Some researchers have reported losses around 7%–40% of stored bean seeds [31,37], which equates to a loss of 1.59–9.12 million tons annually in the world caused by this bruchid [38]. On the other hand, the raspberry weevil, Aegorhinus superciliosus (Guérin) (Coleoptera: Curculionidae), is a significant pest of fruit trees such as European hazelnut (Corylus avellana L.) (Betulaceae), blueberry (Vaccinium corymbosum L.) (Ericaceae), raspberry (Rubus idaeus L.) (Rosaceae) and other minor fruit trees. The larvae of this weevil attack the plant’s root system, boring into the main root, affecting water and nutrient uptake and causing the death of the plant. Moreover, adult weevils feed on the leaves and shoots of the season, affecting vegetative growth [39,40]. Currently, broad-spectrum synthetic pesticides such as pyrethroids and organophosphates are the most used to control these pests [41,42]. However, their application on stored dry beans and fruit orchards has increased public concern over pesticide safety and environmental damage [43–46]. In this respect, our study’s aim is to promote research with endemic plant species in Chile as a natural alternative more compatible with safer pest control approaches. Hence, we assessed the insecticidal potential of canelo essential oil as a toxicological agent against A. obtectus, a stored dried bean pest and as a repellent against A. superciliosus, a fruit tree pest, and we determined the D. winteri EO chemical profile by gas chromatography coupled to mass spectrometry (GC-MS). Insects 2020, 11, 335 3 of 15 2. Materials and Methods 2.1. Plant Material and Essential Oil Extraction Drimys winteri aerial parts were collected in the fall of 2017 from Vilcún (38◦40’08.1” S, 72◦16’22.9” W), La Araucanía, Chile. Its identity was confirmed by comparing macroscopic and microscopic morphologic characteristics to Chilean flora and specimens in the herbarium at the Universidad de Concepción, Chile. Fresh leaves and shoots were washed with distilled water to remove any residue. The extraction of the essential oil from leaves and shoots of canelo—hereafter referred to as CEO—was performed according to Zapata and Smagghe [27]. Briefly, chopped leaves and shoots of D. winteri (0.4 kg) were subjected to hydrodistillation for 4 h in a Clevenger apparatus. Then, the CEO was dried over anhydrous sodium sulfate [47,48]. 2.2. Essential Oil Analysis The CEO was analyzed with gas chromatography coupled to mass spectrometer (GC/MS), using the following instrumentation: a Thermo Focus GC (Thermo Fisher Scientific, Waltham, MA, USA) coupled to a Thermo DSQ quadrupole mass spectrometric detector with an integrated data system (Xcalibur 2.0, Thermo Fisher Scientific, Inc., Waltham, MA, USA). One microliter aliquot of the CEO 1 diluted in hexane was injected at a concentration of 1 µg µL− in a capillary column BPX5 (30-m length, 0.25-µm film thickness, 0.25-mm inner diameter, SGE Forte, Trajan Scientific and Medical, Ringwood, × Victoria, Australia) in splitless mode. The operating conditions were on-column injection: injector temperature of 250 ◦C, transfer line temperature of 250 ◦C; detector temperature of 250 ◦C; carrier gas: 1 He at 1.0 mL min− , oven temperature program: 40 ◦C for 2 min, increased to 250 ◦C at 5 ◦C/min, followed by 250 ◦C for 5 min. The mass spectra were obtained at an ionization voltage of 70 eV. Recording conditions employed a scan time of 1.5 s and a mass range of 30–400 amu. The compounds were identified based on the comparisons of the mass spectra with a library database (NIST ver.